Behavioral Prevention, Treatment, and Rehabilitation of Using Western and Chinese Medicines or Herbal Products among the Public in Response to COVID-19 in Hong Kong: A Cross-Sectional Study.

The coronavirus disease 2019 (COVID-19) pandemic occurred in Hong Kong for more than two years. This article conducted a cross-sectional study for participants to investigate the behavioral prevention, treatment, and rehabilitation of using Western medicines or herbal products for COVID-19 in Hong Kong. A questionnaire was designed and performed over 2 weeks from 1 May to 15 May 2022. It consisted of five parts with around 20 questions conducted including sociodemographic information, prevention, treatment, rehabilitation of COVID-19, and also the sources of information. The pattern usage of Chinese or Western medicines for COVID-19 was studied after data collection. 318 people participated in this survey, and only 311 were qualified. The sociodemographic information, e.g., personal educational level, and behavior for the prevention of COVID-19, which included wearing masks (98.7%), using alcohol hand sanitizer (83.0%), washing hands frequently (82.4%), avoiding crowds (53.1%), and staying home more often (50.6%). Western medicines, such as antipyretic drugs, antitussive drugs, and pain reliever drugs, whilst Chinese medicines, such as Lianhua Qingwen Jiaonang, Huoxiang Zhengqi San or Wan, and Nin Jiom Pei Pa Koa, were most commonly used in the treatment and rehabilitation periods of COVID-19. Herbal products, including lemon, honey, ginger, and herbal tea, were used as a daily diet to fight against COVID-19. Based on the result findings, Chinese medicines or herbal products were used during the COVID-19 pandemic, but most of the participants used an unknown Chinese medicine practitioner's prescription and self-administered Chinese medicine. The pattern of Chinese medicines and Western medicines' usage in the prevention, treatment, and rehabilitation of COVID-19 was also investigated; this showed a statistically significant association between the variables according to gender, age, and Chinese or Western medicines for further investigation.

A Novel Probiotic-Based Oral Vaccine against SARS-CoV-2 Omicron Variant B.1.1.529.

COVID-19 pandemic, caused by the SARS-CoV-2 virus, is still affecting the entire world via the rapid emergence of new contagious variants. Vaccination remains the most effective prevention strategy for viral infection, yet not all countries have sufficient access to vaccines due to limitations in manufacturing and transportation. Thus, there is an urgent need to develop an easy-to-use, safe, and low-cost vaccination approach. Genetically modified microorganisms, especially probiotics, are now commonly recognized as attractive vehicles for delivering bioactive molecules via oral and mucosal routes. In this study, Lactobacillus casei has been selected as the oral vaccine candidate based on its' natural immunoadjuvant properties and the ability to resist acidic gastric environment, to express antigens of SARS-CoV-2 Omicron variant B.1.1.529 with B-cell and T-cell epitopes. This newly developed vaccine, OMGVac, was shown to elicit a robust IgG systemic immune response against the spike protein of Omicron variant B.1.1.529 in Golden Syrian hamsters. No adverse effects were found throughout this study, and the overall safety was evaluated in terms of physiological and histopathological examinations of different organs harvested. In addition, this study illustrated the use of the recombinant probiotic as a live delivery vector in the initiation of systemic immunity, which shed light on the future development of next-generation vaccines to combat emerging infectious diseases.

Characterization of the Gut Microbiome in Healthy Dogs and Dogs with Diabetes Mellitus.

With a close pathogenetic resemblance to human diabetes, canine Diabetes Mellitus, a chronic metabolic disease featuring abnormally high blood sugar levels, is increasing in prevalence worldwide. Unlike humans, canine glycemic control requires life-long insulin injections and dietary control in most cases, thereby jeopardizing diabetic dogs' quality of life and increasing the difficulty of disease control. While many research studies have focused on elucidating the relationship between the canine gut microbiome and diseases, there is currently no research on the subject of diabetes mellitus in dogs. We hypothesized that the gut microbiome of canines with diabetes mellitus is different from that of healthy controls. Thus, we performed targeted 16S rRNA sequencing and comprehensive bioinformatic analysis to compare the gut microbiome profiles of 16 diabetic dogs with those of 32 healthy dogs. Clostridioides difficile, Phocaeicola plebeius, Lacrimispora indolis, and Butyricicoccus pullicaecorum were found to be enriched in diabetic dogs. A distinct shift towards carbohydrate degradation metabolic pathways was found to be differentially abundant in the diabetic subjects. Alteration of the co-occurrence network was also evident in the diabetic group. In conclusion, our study suggests that the gut microbial landscape differs in diabetic canines at the genera, species, functional, and network levels. These findings have significant implications for disease management, and thus warrant further research.

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 ß-Agonists and Analogues.

Immunoassay methods are important for monitoring ß-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various ß-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 ß-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of ß-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

Investigation of association of chemical profiles with the tracheobronchial relaxant activity of Chinese medicinal herb Beimu derived from various Fritillaria species.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae Bulbus (Beimu in Chinese) is derived from the bulbus of many Fritillaria species (family Liliaceae), which has been used as an antitussive herb in traditional Chinese medicine for more than 2000 years. Due to the complexity of plant origins and significant variations in chemical profiles, the characterization of the profile of the major bioactive constituents and its association with pharmacological activity are important for the quality control of Beimu herbs from different origins. AIM OF THE STUDY: This study aims to investigate the distribution of major bioactive isosteroidal alkaloids in Beimu herbs of different origins and its correlation with the tracheobronchial relaxant activity. METHODS: Quantification of 7 main bioactive 5α-cevanine isosteroidal alkaloids, including ebeiedine, ebeiedinone, hupehenine, isoverticine, verticine, verticinone and imperialine, in 23 Fritillaria species was performed using gas chromatography. The relaxant effect of different extracts of 4 commonly used Beimu herbs, namely Zhe-Beimu (F. thunbergii Miq.), Chuan-Beimu (F. cirrhosa D. Don), Hubei-Beimu (F. hupehensis Hsiao et K. C. Hsia) and Yi-Beimu (F. pallidiflora Schrenk), was evaluated using rat isolated tracheal and bronchial preparations pre-contracted with carbachol, the well established in vitro antitussive model. RESULTS: Amongst 23 Fritillaria species detected, significant variations of the types and quantities of 7 major isosteroidal alkaloids were determined, which served as an important indicator for the classification of different Beimu herbs with distinct geographic distributions. Based on the type and quantity of these alkaloids, different origins of Beimu could be clearly clustered into several subgroups by principal component analysis. Furthermore, both crude alkaloid and water extracts of all 4 Beimu herbs showed a dose-dependent tracheobronchial relaxation with different potencies. The total content of alkaloids (weight adjusted based on the activity of individual alkaloids) in Beimu extracts significantly correlated with their tracheobronchial relaxation effects (r2 > 0.9, p < 0.001). CONCLUSIONS: The results demonstrated that the differences in chemical profile of major bioactive isosteroidal alkaloids and pharmacological activity of Beimu could be incorporated into a simple and unified method for quality control and potential prediction of activity of Beimu herbs from different origins.

Cardiovascular Protective Effects of Salvianic Acid A on db/db Mice with Elevated Homocysteine Level.

The onsets of left ventricular hypertrophy (LVH) and endothelial dysfunction (ED) in diabetics, especially in those with elevated homocysteine (Hcy), precede the development of cardiovascular (CV) events. Salvianic acid A (SAA) is a renowned Traditional Chinese Medicine (TCM) that has been applied in the treatment of cardiovascular disease for many decades. In this study, we aimed (1) to investigate the CV protective effects of SAA on ameliorating LVH and ED in db/db mice with elevated blood Hcy level and (2) to decipher whether the observed CV protective effects of SAA are associated with Hcy metabolism by modulating the methylation potential and redox status in the liver of the db/db mice with elevated blood Hcy level. Our results found that the administration of SAA could significantly slow down the build-up of left ventricular mass and ameliorate ED. Immunological assay analysis on the mouse liver tissue also indicated that SAA treatment on db/db mice with elevated Hcy was associated with reduced methylation potential but improved redox status. In conclusion, we revealed that SAA has the potential to protect against the hyperglycemia- and hyperhomocysteinemia-induced oxidative stress on diabetic mice via modulation in Hcy metabolism.

Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 µM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 µM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.

Discovery of a ROCK inhibitor, FPND, which prevents cerebral hemorrhage through maintaining vascular integrity by interference with VE-cadherin.

Hemorrhagic stroke occurs when a weakened vessel ruptures and bleeds into the surrounding brain, leading to high rates of death and disability worldwide. A series of complex pathophysiological cascades contribute to the risk of hemorrhagic stroke, and no therapies have proven effective to prevent hemorrhagic stroke. Stabilization of vascular integrity has been considered as a potential therapeutic target for hemorrhagic stroke. ROCKs, which belong to the serine/threonine protein kinase family and participate in the organization of actin cytoskeleton, have become attractive targets for the treatment of strokes. In this study, in vitro enzyme-based assays revealed that a new compound (FPND) with a novel scaffold identified by docking-based virtual screening could inhibit ROCK1 specifically at low micromolar concentration. Molecular modeling showed that FPND preferentially interacted with ROCK1, and the difference between the binding affinity of FPND toward ROCK1 and ROCK2 primarily resulted from non-polar contributions. Furthermore, FPND significantly prevented statin-induced cerebral hemorrhage in a zebrafish model. In addition, in vitro studies using the xCELLigence RTCA system, immunofluorescence and western blotting revealed that FPND prevented statin-induced cerebral hemorrhage by enhancing endothelial cell-cell junctions through inhibiting the ROCK-mediated VE-cadherin signaling pathway. As indicated by the extremely low toxicity of FPND against mice, it is safe and can potentially prevent vascular integrity loss-related diseases, such as hemorrhagic stroke.

Chemical Analysis of Astragali Complanati Semen and Its Hypocholesterolemic Effect Using Serum Metabolomics Based on Gas Chromatography-Mass Spectrometry.

The hypocholesterolemic protective effect of the dried seed of Astragalus complanatus (ACS) was investigated in rats fed with normal diet, high cholesterol diet (HCD), and HCD plus 70% ethanol extract of ACS (600 mg/kg/day) by oral gavage for four weeks. ACS extract was tested to be rich in antioxidants, which may be contributed to its high content of phenolic compounds. Consumption of ACS remarkably suppressed the elevated total cholesterol (p < 0.01) and LDL-C (p < 0.001) induced by HCD. Chemical constituents of ACS extract were analyzed by ultra-performance liquid chromatography coupled with electrospray ionization orbitrap mass spectrometry and the results showed that the ACS extract mainly consisted of phenolic compounds including flavonoids and flavonoid glycosides. In addition, based on the serum fatty acid profiles, elucidated using gas chromatography-mass spectrometry, free and esterified fatty acids including docosapentaenoic acid, adrenic acid, dihomo-γ-linolenic acid and arachidonic acid were regulated in ACS treatment group. Western blot results further indicated the protein expression of peroxisome proliferator-activated receptor alpha (PPARα) (p < 0.05) in liver was upregulated in ACS treatment group. To conclude, our results clearly demonstrated that ACS provides beneficial effect on lowering HCD associated detrimental change.

Production of polyhydroxyalkanoates (PHA) using sludge from different wastewater treatment processes and the potential for medical and pharmaceutical applications.

In this study, seven strains of bacteria with polyhydroxyalkanoates (PHA)-producing ability (i.e. Bacillus cereus, Pseudomonas putida, Bacillus pumilus, Pseudomona huttiensis, Yersinia frederiksenii, Aeromonas ichthiosmia, and Sphingopyxis terrae) were isolated from various waste treatment plants in Hong Kong. Simultaneous wastewater treatment and PHA accumulation were successfully achieved in the bioreactors using isolated bacteria from different sludges. At the organic loading less than 13,000 ppm, more than 95% of chemical oxygen demand (COD) was removed by the isolated strains before the decrease of PHA accumulation. In addition, more than 95% of nitrogen removal was achieved by all isolated strains. In the bioreactors inoculated with single strains, the highest yields of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyvalerate) (PHV) were obtained in A. ichthiosmia (84 mg PHB/g) and B. cereus (69 mg/g), respectively. For the mixed culture, the highest yields of PHB and PHV were increased by 55% and 45% in the system inoculated with B. pumilus and A. ichthiosmia. The biologically synthesized PHA also showed the potential applications in drug delivery and tissue engineering. PHA-nanoparticles loaded with pyrene were successfully prepared by recombinant Escherichia coli. The results of in vitro drug release and biocompatibility tests revealed that nanoparticles could be used as safer dray carriers with high loading capacity and efficiency. After 20 days, the cells successfully grew on 90% of the PHA-aortic valve.

Chemical and pharmacological evaluations on the extract of Scutellaria baicalensis Georgi (Huang-Qin) prepared by various extraction methods.

BACKGROUND: This study reported a comprehensive approach (comparing the extraction yields, chemical profiles, antioxidant properties and CYP450-inhibitory effects) to evaluated the effectiveness of various extraction methods [microwave-assisted extraction using water (MAE-W), heat reflux extraction using water (HRE-W), ultrasonic extraction using 70 % ethanol and ultrasonic extraction using ethanol (UE-E)] for Huang-Qin (HQ), the dried root of Scutellaria baicalensis Georgi. RESULTS: The HQ extraction efficiency by MAE-W was the best. The chemical profiles of extracts obtained using HRE-W and MAE-W were similar; whereas more flavones but less flavone glycosides were detected in the UE-E extract. There was no difference in the antioxidant properties among different extracts. In vitro human liver microsome assays illustrated that all extracts possessed herb-drug interaction potentials but the UE-E extract are shown with a potent interaction with CYP3A4-metabolized drugs. CONCLUSION: MAE-W is a favorable method for the preparation of HQ extracts based on extraction yield, pharmacological properties and safety.

Inhibition of human equilibrative nucleoside transporters by 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine.

Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.

Novel cycloartane triterpenoid from Cimicifuga foetida (Sheng ma) induces mitochondrial apoptosis via inhibiting Raf/MEK/ERK pathway and Akt phosphorylation in human breast carcinoma MCF-7 cells.

BACKGROUND: Cycloartane triterpenoids exhibited anticancer effects. This study aims to identify any potential novel anticancer cycloartane triterpenoids from Cimicifuga foetida L. rhizome (Sheng ma) and the mode of actions. METHODS: Cycloartane triterpenoids were isolated from the C. foetida rhizome by a series of column chromatography and identified by IR, MS and NMR. Their anticancer effects on several human cancer cell lines, MCF-7, HepG2, HepG2/ADM, HeLa, and PC3, and normal human mammary epithelial cells MCF10A were investigated by colony formation and MTT assays. Morphological analysis of apoptosis induction was performed by acridine orange/ethidium bromide dual-staining and Hoechst 33258 nuclear staining. The cell-cycle profile and annexin V staining were evaluated by flow cytometry. Apoptosis were investigated by measuring changes in mitochondrial membrane potential and analyzing expression of cell cycle- and apoptosis-related proteins in MCF-7 cells by Western blotting. RESULTS: A novel cycloartane triterpenoid, 25-O-acetyl-7,8-didehydrocimigenol-3-O-ß-d-(2-acetyl)xylopyranoside (ADHC-AXpn), together with the known 7,8-didehydrocimigenol-3-O-ß-d-xylopyranoside (DHC-Xpn) were isolated. MCF-7 growth was significantly inhibited by ADHC-AXpn in a dose- and time-dependent manner (IC50: 27.81 µM at 48 h; P = 0.004 vs. control at 25 µM for 48 h treatment), and ADHC-AXpn was selectively cytotoxic for cancerous cells (MCF-7, HepG2/ADM, HepG2 and HELA cells) based on its higher IC50 values for normal cells MCF10A (IC50: 78.63 µM at 48 h) than for tumor cells. In MCF-7 cells, ADHC-AXpn induced G2/M cell cycle arrest by mediating cyclin-B1, and CDK1 and its phosphorylation; and induced apoptosis through the mitochondrial-mediated apoptotic pathway, with inhibition of Akt activation. As ADHC-AXpn suppressed phosphorylation of ERK1/2, Raf and Akt proteins in MCF-7 cells, its apoptotic effect might be associated with Raf/MEK/ERK signaling and Akt activation. CONCLUSIONS: ADHC-AXpn significantly suppressed the growth of MCF-7 cells, induced mitochondrial apoptosis and cell-cycle arrest, and inhibited Raf/MEK/ERK signaling pathway and Akt phosphorylation.

Vascular Contributions to Cognitive Impairment and Treatments with Traditional Chinese Medicine.

The prevalence of cognitive impairment and dementia caused by cerebrovascular disease is likely to increase with the global aging population. Vascular contributions to cognitive impairment and dementia (VCID) is a wide spectrum term used to include a diverse heterogeneous group of cognitive syndromes with vascular factors regardless of the cause of pathogenesis. VCID ranges from mild cognitive impairment to full-blown dementia with vascular dementia (VaD) as the most severe stage. It is further complexed by the coexistence of other forms of dementia such as Alzheimer's disease (AD). Recent researches in the functions of the neurovascular unit (NVU) suggest that dysfunction of the NVU might be the cause of primary vascular events in the brain that leads to further neurodegeneration. In this review, we have briefly summarized various forms of VCID. There is currently no standard therapy for VCID or dementia. Given the fact that Traditional Chinese Medicine (TCM) has gained popularity worldwide, we also reviewed recent scientific and clinical findings on various antidementia TCM for the treatment of VCID, including Salvia miltiorrhiza, Huperzia serrata, Ligusticum chuanxiong, Ginkgo biloba, Panax ginseng, and also TCM formula Sailuotong capsule (SLT) and Fufangdanshen tablets (FFDS).

Effect of Hedyotis diffusa water extract on protecting human hepatocyte cells (LO2) from H2O2-induced cytotoxicity.

CONTEXT: Natural products are good sources of natural dietary antioxidants that are believed to protect the body against hepatotoxic effect induced by oxidative stress. Hedyotis diffusa Willd (Rubiaceae) (HDW) is a traditional Chinese medicinal herb that has been shown to possess a variety of antioxidant properties. OBJECTIVE: The present study examines and explains the cell protective property of HDW water extract (WEHDW). MATERIALS AND METHODS: 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay was used to measure the free radical scavenging property of WEHDW (0.001-10 mg/mL). The protective effect of WEHDW (0.3-10 mg/mL 2 h pretreatment) against hydrogen peroxide (H2O2, 200 µM for 6 h) induced cytotoxicity in human hepatic cells, LO2, was evaluated using cell viability assay and nuclear staining. The molecular pathway of WEHDW's effect was investigated by using Western blot assay. RESULTS: WEHDW had a 50% scavenging concentration (SC50) at 0.153 mg/mL in the DPPH assay. Exposure of LO2 cells to H2O2 resulted in apoptosis which could be markedly attenuated by pre-treating WEHDW in a concentration-dependent manner (0.5, 1, 3, 5, or 10 mg/mL) (all with p < 0.001, versus control). Moreover, Hoechst (nuclear) staining showed that 1 mg/mL WEHDW could protect LO2 cells by attenuating apoptotic cell death mediated by H2O2. It was found that WEHDW reversed H2O2-induced activation of MEK/ERK pathway and H2O2-induced inhibition of P13-K/AKT/GSK3ß pathway in LO2 cells. DISCUSSION AND CONCLUSION: WEHDW may help to improve the antioxidant defense system, resulting in prevention of oxidative stress-related fatty liver diseases.

Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

In vitro vitamin K(2) and 1α,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice.

In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.

Cortex Fraxini (Qingpi) Protects Rat Pheochromocytoma Cells against 6-Hydroxydopamine-Induced Apoptosis.

Parkinson's disease (PD) is a chronic neurodegenerative disorder having close relationship with oxidative stress induced by reactive oxygen species (ROS). Cortex Fraxini (QP) is a kind of traditional Chinese medicinal herb with antioxidant properties. It may be a potential candidate for preventing the development of chronic neurodegenerative diseases. Thus, the key objective of the current study was to investigate the neuroprotective effect of QP water extract on 6-hydroxydopamine (6-OHDA) induced apoptosis in rat pheochromocytoma (PC12) cells. It was found that QP water extract possesses strong antioxidant property with SC50 = 0.15 mg/mL. Total phenolic content of QP water extract was found to be 200.78 ± 2.65 mg GAE/g. QP water extract's free radical scavenging capacity was demonstrated by reversing the increased level of intracellular ROS induced by 6-OHDA, using 2',7'-dichlorodihydrofluorescein diacetate. Moreover, QP water extract (0.5 mg/mL) could remarkably increase the viability of PC12 cells treated with 6-OHDA. The protective effect of QP water extract was found to be via inhibiting MEK/ERK pathway and reversing PI3-K/Akt/GSK3ß pathway. The current results suggest that QP might be a potential candidate for preventing the development of neurodegenerative diseases, such as PD.

Centipeda minima (Ebushicao) extract inhibits PI3K-Akt-mTOR signaling in nasopharyngeal carcinoma CNE-1 cells.

BACKGROUND: Centipeda minima (Ebushicao) has been used for the treatment of various diseases, such as nasal allergies, rhinitis and sinusitis, nasopharyngeal carcinoma, cough, and headache. This study aims to investigate the anticancer activities of Centipeda minima ethanol extracts (CME) against nasopharyngeal carcinoma cell CNE-1 and their underlying mechanism. METHODS: CNE-1 cells were treated with different concentrations (15-50 µg/mL) of CME for different time intervals (24, 48, and 72 h). Cytotoxicity of CME was determined by MTT assay. Cell morphological changes were observed by fluorescence microscopy after HO 33258 staining. Cell cycle status was evaluated by flow cytometry following propidium iodide staining. Apoptosis was detected by flow cytometry following annexin V-FITC/PI staining. The levels of apoptosis-associated and PI3K-Akt-mTOR signaling related proteins were measured by western blotting analysis. RESULTS: CME (15-50 µg/mL) significantly inhibited the proliferation of CNE-1 in a dose- and time-dependent manner (P = 0.026 for 15 µg/mL, P < 0.001 for 25, 30, 40, and 50 µg/mL, respectively); the IC50 values (µg/mL) were 41.57 ± 0.17, 30.34 ± 0.06 and 24.98 ± 0.08 for 24, 48 and 72 h treatments, respectively. Significant morphological changes of CNE-1 cells displaying apoptosis were observed after CME treatment. CME showed low cytotoxicity toward normal LO2 cells. CNE-1 cells were arrested in the G2/M phase while treated with 15, 25, 40 µg/mL of CME, respectively (P = 0.032, P = 0.0053, P < 0.001). CME (15, 25, 40 µg/mL) down-regulated Bcl-2 expression (P = 0.032, P = 0.0074, P < 0.001), and up-regulated Bax (P = 0.026, P = 0.0056, P < 0.001) with activation of caspase-3, caspase-8, caspase-9, and PARP observed in CNE-1 cells (P = 0.015, P = 0.0067, P < 0.001 for caspase 3; P = 0.210, 0.028, < 0.001 for caspase 8; P = 0.152, 0.082, 0.0080 for caspase 9; P = 0.265, 0.0072, < 0.001 for PARP). CME suppressed the activation of the PI3K-AKT-mTOR pathway (P = 0.03, 0.0007, 0.004, 0.006, 0.022 for p-PI3K, p-Akt-Ser(473), p-Akt-Thr(308), p-mTOR-Ser(2448), p-mTOR-Ser(2481), respectively after 40 µg/mL of CME treated for 24 h). CONCLUSION: CME inhibited the proliferation of CNE-1 cells and activation of the PI3K-AKT-mTOR signaling pathway.

A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo.

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 µM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 µM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 µM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.